Members of the hedgehog family of signaling molecules mediate many important short- and longrange patterning processes during invertebrate and vertebrate embryonic, fetal, and adult development. In Drosophila melanogaster, a single hedgehog gene regulates segmental and imaginal disc patterning. In contrast, in vertebrates, a hedgehog gene family (e.g., in mammals, Shh, Dhh, Ihh, collectively “Hh”) is involved in the control of proliferation, differentiation, migration, and survival of cells and tissues derived from all three germ layers, including, e.g., left-right asymmetry, CNS development, somites and limb patterning, chondrogenesis, skeletogenesis and spermogenesis.
Hedgehog signaling occurs through the interaction of hedgehog protein with the hedgehog receptor, Patched (Ptch), and the co-receptor Smoothened (Smo). There are two mammalian homologs of Ptch, Ptch-1 and Ptch-2 (“collectively “Ptch”), both of which are 12 transmembrane proteins containing a sterol sensing domain (Motoyama et al., Nature Genetics 18: 104-106 (1998), Carpenter et al., P.N.A.S. (U.S.A.) 95 (23): 13630-40 (1998). The interaction of Hh with Ptch triggers a signaling cascade that results in the regulation of transcription by zinc-finger transcriptions factors of the Gli family.
Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin. 43:7 (1993)). Cancer features one of more of the following characteristics: (1) an the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, (2) the invasion of adjacent tissues by these neoplastic tumor cells, and (3) the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
As often is the case when pathways that are active during embryogenic development and mostly inactive in adults, reactivation of hedgehog signaling has been implicated in a wide variety of cancers and carcinogenesis. The earliest examples of Hh signaling in cancers came from the discovery that Gorlin's syndrome, in which patients frequently suffer basal cell carcinomas and are also predisposed to medulloblastomas and rhabdomysocarcomas, is due to an inactivating mutation in Ptch, resulting in Hh pathway activation (Hahn et al 1998 Cell 85 p841; Johnson et al 1996, Science 272 p1668). Subsequently inactivating mutations in Ptch (˜90%) and or activating mutations in Smo (˜10%) were found to be responsible for sporadic basal cell carcinomas (Xie et al 1998, Nature 391 p90).
Recently, it has become clear that another class of Hh-associated cancers exist, which depend on Hh ligand secretion from the tumor rather than mutational activation for pathway activation. Such cancers include prostate, pancreatic and small cell lung cancers (Watkins et al 2003, Nature 422 p313; Thayer et al 2003 Nature 425 p851; Berman et al 2003 Nature 425 p846). A subset of such cancers can be treated by Hh antagonists such as small molecule antagonists of Smo or anti-Hh antibody 5E1 (Chen et al 2002, PNAS 99 p14071; Williams et al 2003 PNAS 100 p4616; Rubin and de Sauvage 2006 Nature Reviews Drug Discovery 5 p1026). While not all Hh-expressing tumors respond to such antagonists, it is very likely that those that do not express Hh will not respond; indeed the Hh-negative DLD-1 colorectal xenograft model is not inhibited by such treatment under conditions where Hh-positive LS180, HT29 and HT55 tumor models are (Yauch/de Sauvage et al. January 2008). As a result, there is a need for an effective technique for determining hedgehog expression prior to application of the hedgehog antagonists so as to identify hedgehog-secreting tumors, in order to maximize the overall response rate.
Currently available antibodies that bind to mammalian hedgehog (e.g., H160, Santa Cruz Biotech) are ineffective reagents to detect the presence of hedgehog signaling because they do not show sufficient sensitivity in the absence of background staining This is particularly true on FFPE (formalin-fixed paraffin-embedded) tissue specimens.
As a result, there is a need for antibodies that bind to mammalian hedgehog (e.g., sonic hedgehog, Indian hedgehog and desert hedgehog), particularly in FFPE specimens, for use to detecting the expression of hedgehog both in diagnostic assays and treatment regimens.